1. Field of the Invention
The invention relates to retrovirus vectors for use in the expression of recombinant proteins and peptides. In particular, the invention relates to a method for producing retrovirus vectors using separate genes for the gag, pol and env structural retrovirus genes.
2. History of the Prior Art
Retroviruses which can accept and express foreign genes (e.g., the Moloney murine leukemia virus (MoMLV)) are useful in a number of applications, including gene therapy. However, since recombinant retroviruses are defective, they require assistance in order to produce infectious vector particles. This assistance can be provided by using helper virus carrying packaging cell lines that include structural genes of the retrovirus (e.g., env, gag and pol) under the control of regulatory sequences.
The use of retroviral vectors to deliver encoded material to a host raises the possibility that recombination of the genes provided by the vector virus and helper virus can occur to produce a replication-competent virus capable of infecting the host with viral proteins. Separation of the helper virus gag/pol and env encoding genes onto two separate plasmids in the packaging cell line (one which codes for the viral gag/pol proteins and another which does for the viral envelope protein) makes this possibility more remote by requiring that at least two recombination events occur for a replication-competent retrovirus (RCR) to be produced. Removal of the "psgr" packaging sequence from the plasmids also helps to reduce the risk of RCR infection. This arrangement significantly decreases, but does not eliminate, the incidence of RCR production by retrovirus packaging cell lines.
The invention is directed to a method for producing replication-incompetent retroviral (RIR) vectors. The vectors are produced according to the invention using packaging cell lines in which the helper virus gag, pol and env genes are each separated onto different provirus plasmids; i.e., one which codes for the viral gag protein, another which codes for the viral pol protein and a third which codes for the viral envelope protein.
To this end, the invention provides provirus plasmids which separately code for gag, pol and envelope proteins (respectively, pGag, pPol and pEnv), as well as a packaging cell line transfected with the plasmids for use in producing RIR vectors.
RIR vector products of the method of the invention are also provided by the invention. In one aspect, such RIR products are formed from a packaging cell line in which pGag and pPol provirus plasmids code for viral proteins from the same parent retrovirus.
In another aspect, RIR products are formed from a packaging cell line in which the pGag and pPol provirus plasmids code for viral proteins from different parent retroviruses.
The invention also provides packaging cell lines in which expression of one or more of the gag, pol and envelope proteins is enhanced by the addition of, respectively, pGag and pPol.